Thrombotic thrombocytopenic

Flora Peyvandi Angelo Bianchi Bonomi Hemophilia and Center IRCCS Ca’ Granda Ospedale Maggiore Policlinico University of Milan Milan, Italy Disclosures

Research Support/P.I. No relevant conflicts of interest to declare

Employee No relevant conflicts of interest to declare

Consultant Kedrion, Octapharma

Major Stockholder No relevant conflicts of interest to declare

Speakers Bureau Shire, Alnylam

Honoraria No relevant conflicts of interest to declare Scientific Advisory Ablynx, Shire, Roche Board Objectives

• Advances in understanding the pathogenetic mechanisms and the resulting clinical implications in TTP

• Which tests need to be done for diagnosis of congenital and acquired TTP

• Standard and novel therapies for congenital and acquired TTP

• Potential predictive markers of relapse and implications on patient management during remission Thrombotic (TTP)

First described in 1924 by Moschcowitz, TTP is a thrombotic microangiopathy characterized by:

• Disseminated formation of - rich thrombi in the microvasculature → Tissue with neurological, myocardial, renal signs & symptoms • consumption → Severe • fragmentation → Hemolytic anemia TTP epidemiology

• Acute onset • Rare: 5-11 cases / million people / year • Two forms: congenital (<5%), acquired (>95%) • M:F ratio 1:3 • Peak of incidence: III-IV decades • Mortality reduced from 90% to 10-20% with appropriate therapy • Risk of recurrence: 30-35%

Peyvandi et al, Haematologica 2010 TTP clinical features

Bleeding 33 patients with ≥ 3 acute episodes + Thrombosis

“Old” diagnostic pentad: • Microangiopathic hemolytic anemia • Thrombocytopenia • Fluctuating neurologic signs • Fever • Renal impairment

ScullyLotta et et al, al, BJH BJH 20122010 TTP pathophysiology

• Caused by ADAMTS13 deficiency (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13) • ADAMTS13 cleaves the VWF subunit at the Tyr1605–Met1606 peptide bond in the A2 domain

Furlan M, et al. Blood 1996; Tsai HM. Blood 1996; Zheng XL, et al. JBC 2001; Levy GG, et al. Nature 2001; Fujikawa K, et al. Blood 2001, Kremer Hovinga et al, Nat Rev Dis Primers 2017 ADAMTS13 deficiency

ADAMTS13 deficiency normal values 40-160% severe deficiency <10%

Congenital Acquired (<5%) (>95%)

ADAMTS13 gene Anti-ADAMTS13 mutations autoantibodies TTP diagnosis flowchart

1) ADAMTS13 activity to confirm TTP Clinical diagnosis of TTP clinical diagnosis

2) Anti-ADAMTS13 IgG to investigate ADAMTS13 severe the cause of ADAMTS13 deficiency deficiency (<10%) 3) Sequencing of ADAMTS13 gene in selected cases Anti-ADAMTS13 IgG

Positive Negative

Acquired TTP Congenital TTP (95-98%) (2-5%) ADAMTS13 assays

• ADAMTS13 activity • ADAMTS13 antigen • Anti-ADAMTS13 antibodies – Total anti-ADAMTS13 IgG – Neutralizing ADAMTS13 activity (mixing assay) – ADAMTS13-specific immune complexes ADAMTS13 activity assays

…and more

Peyvandi et al, JTH 2010 FRETS-VWF73 assay

• Gold standard • Based on FRET (fluorescence resonance energy transfer) mechanism • Relatively fast (although at least 2 hours) • Easy to perform (although not automated)

ADAMTS13 absentpresentNOemission emissionat 440at 440 nm nm

ADAMTS13 Kokame et al, BJH 2005 Anti-ADAMTS13 antibodies

• ELISA-based methods detecting anti-ADAMTS13 IgG are the most used (developed in-house, commercial kits)

HRP substrate Streptavidin HRP-conjugate 2 Patient’s plasma Biotinylated anti-human IgG

Anti-ADAMTS13 antibody (in 1 patient’s plasma) Cell-colture conditioned media rADAMTS13-V5 containing ADAMTS13 Anti-V5 antibody

Anti-mouse IgG

In-house anti-ADAMTS13 IgG ELISA set-up (Bettoni et al, JTH 2012) TTP treament: rationale

ADAMTS13 deficiency normal values 40-160% severe deficiency <10%

Congenital Acquired (<5%) (>95%)

Replace functional ADAMTS13 Remove anti-ADAMTS13 antibodies Replace functional ADAMTS13 Down-regulate immune system activation Current and novel therapies: congenital TTP

Current therapies • (15-20 ml/Kg/die) • Virally inactivated factor VIII concentrate containing ADAMTS13 Novel therapies • Recombinant ADAMTS13 (rADAMTS13) • Gene therapy • Anfibatide

Allford et al, BJH 2000; Scully et al, BJH 2006; Kopic et al, JTH 2016; Scully et al, Blood 2016 128:135, ASH CONGRESS 2016; Jin et al, Blood 2013; Laje et al, Blood 2009; Verhenne et al, ATVB 2017; Zheng et al, Blood Adv 2016 rADAMTS13

• In contrast to plasma infusions, rADAMTS13 can be infused at lower volumes and contains no additives of human or animal origin

• SHP655 (former BAX930), Shire (former Baxalta)

• Preclinical and phase I clinical trial (NCT02216084) completed: – 15 patients, three dosing cohorts – rADAMTS13 was safe and well tolerated – Immunogenicity tests performed at screening, pre-dose and upon study completion, were negative in all subjects

• Phase 3 trial with SHP655 to be started (randomized, open-label, 2-period crossover study)

rADAMTS13 may provide an important alternative replacement therapy for patients with congenital TTP

Kopic et al, JTH 2016; Scully et al, Blood 2016 128:135, ASH CONGRESS 2016 Gene therapy in animal models

• Transplantation of hematopoietic progenitor cells transduced with a lentiviral vector encoding a full-length ADAMTS13 (activity 26.3±14.3% of normal murine pooled plasma at 5 months after transplantation) resulted in elimination of ultralarge VWF multimers and offered systemic protection against ferric chloride–induced arterial thrombosis Laje et al, Blood 2009

• A single injection of AAV8-based vector expressing N-terminal ADAMTS13 domains (mMDTCS) resulted in long-term expression of functional ADAMTS13 at therapeutic levels (activity 0.6 U/mL at 20 weeks) and prevented TTP in a congenital mouse model of TTP

∼ Jin et al, Blood 2013

• Hydrodynamic tail injection of the nonviral sleeping beauty SB100X transposon system expressing murine ADAMTS13 resulted in long-term expression (25 weeks) of supraphysiological levels of ADAMTS13 transgene (184±17% of normal murine plasma pool) and prevented TTP in a congenital mouse model of TTP Verhenne et al, ATVB 2017 Anfibatide

• Platelet glycoprotein 1b (GPIb) antagonist derived from snake venom • It inhibits platelet agglutination and thrombus formation in vitro and in vivo in murine models of thrombosis (Lei et al, TH 2014)

Platelet counts in ADAMTS13 KO mice treated with PBS or anfibatide (60 ng/g body weight) after shigatoxin challenge

• Blockade of VWF-GP1b interaction by injection of anfibatide twice daily (half-life 5-7 h) was efficacious for treating shigatoxin-induced TTP in ADAMTS13 KO mice

Zheng L et al, Blood Advances 2016 Current and novel therapies: acquired TTP

Current therapies • Plasma exchange • Immunosuppressors Novel therapies • Caplacizumab • Recombinant ADAMTS13 • N-acetylcysteine • Eculizumab • Bortezomib

Scully et al, Br J Hematol 2012 (UK guidelines); Sarode et al, J Clin Apher 2013 (USA guidelines); Scully et al, J Blood Med 2014, Plaimauer et al, JTH 2011 & JTH 2015, Tersteeg et al, ATVB 2015; Jian et al, Blood 2012; Chapin et al, BJH 2012; Tsai et al, BJH 2013; Shortt et al, NEJM 2013; van Balen et al, EJH 2014; Mazepa et al, BJH 2014; Yates et al, Transfusion 2014; Patriquin et al, BJH 2016; Peyvandi et al, NEJM 2016; Peyvandi et al, JTH 2017 Mainstay of TTP treatment for acquired TTP

Standard of care based on two pillars

Daily PEX until confirmed Immunosuppression

Treatement duration platelet normalization (corticosteroids and/or RTX)

Remove ULVWF and anti- ADAMTS13 autoantibodies Replace functional ADAMTS13 Suppress over-activity of immune system

PEX, plasma exchange; RTX, rituximab Scully et al, Br J Hematol 2012 (UK guidelines); Sarode et al, J Clin Apher 2013 (USA guidelines); Scully et al, J Blood Med 2014 Acute phase: plasma exchange

• Gold standard therapy • Reduce mortality from 90% to 10-20%

Rock et al, NEJM 1991

• Start PEX within 24 h • At least a daily plasma volume (PV) exchange (1.5 PV during 1-3 days) • Stop PEX when PLT count >150000/mmc for ≥ 2 consecutive days, and LDH and clinical symptoms recovered • Tapering of PEX may reduce exacerbation

Scully et al, Br J Hematol 2012 (UK guidelines), Sarode et al, J Clin Apher 2013 (USA guidelines) Acute phase: corticosteroids

• Based on clinical practice/case series (Vesely et al Blood 2003, Perotti et al Haematologica 1996, Coppo et al BJH 2006, Cataland et al BJH 2007)

• Associated with PEX • Standard (1 mg/kg/die) versus high dose (10 mg/kg/die for 3 days) methylprednisolone IV • Tapering during 4-8 weeks from remission

Balduini et al, Ann Hematol 2010 Refractory TTP

• Persistent thrombocytopenia or LDH elevation after 7 days, despite daily PEX • Progression of clinical symptoms

• Intensification of PEX every 12 hours, or double plasma volume (Shumak et al, 1995; Bobbio-Pallavicini et al, 1997; Bandarenko & Brecher, 1998; Kahwash & Lockwood, 2004; Nguyen et al, 2008) • Rescue therapy: CSA, VCR, CTX and • Rituximab is the current treatment of choice (Scully et al, BJH 2007) Acute phase: rituximab

• Genetically engineered chimeric murine/human monoclonal antibody directed against the CD20

Indications: • Refractory TTP • Exacerbation (< 30 days from platelet count normalization) • Relapsing chronic TTP (Fakhouri et al, 2005; Scully et al, 2007; Scully et al, 2011) Schedule: • Dosage: 375 mg/mq/week for 4 weeks • When dosed during PEX, postpone next PEX > 4 h later (Hull et al, 2006; Scully et al, 2007) • Intensified regimen (every 3-4 days) during PEX (McDonald et al, 2010) ACUTE PHASE

• Disease duration is variable, clinical response usually achieved after 9-16 days of PEX  may require long hospitalization • PEX takes time, requires special equipment and trained staff • PEX has complications as hypersensitivity reactions, infections, central line infection and thrombosis • Risk of exacerbation • Major thrombotic complications (e.g., , myocardial ) may occur during acute events • Refractoriness to standard treatment • Insufficient immunosuppressive treatment, leading to recurrences  more microvascular thrombosis Still 10% mortality despite PEX and long term consequences of microthrombi formation in the first 3-5 days with loss of memory, and reduction of quality of life

URGENT NEED FOR NEW THERAPIES Caplacizumab

28 kD

• Caplacizumab is a anti-VWF nanobody (Nanobody® is a biologic derived from heavy chain only antibodies ) • Caplacizumab binds to A1 domain of VWF • Immediate inhibition of platelet string formation and consumption of platelets  faster normalisation of platelets  reduction of tissue damage Caplacizumab has not been approved yet The TITAN trial (phase II)

In the Caplacizumab group: • time to a response was significantly reduced (39% reduction in median time, p = 0.005) • Lower number of exacerbations • A lower proportion of patients refractory to treatment

Caplacizumab Placebo Time to response (N=36) (N=39) Median days (95% CI), NO prior PE 3.0 (2.7, 3.9) 4.9 (3.2, 6.6)

Median days (95% CI), one prior PE 2.4 (1.9, 3.0) 4.3 (2.9, 5.7) Overall hazard rate ratio (95% CI) 2.2 (1.3, 3.8) caplacizumab vs. placebo Stratified log-rank test p-value 0.005

Peyvandi et al., NEJM 2016 TITAN trial: major thromboembolic events

A lower proportion of subjects treated with caplacizumab experienced one or more major thromboembolic complications, or died

Peyvandi et al., NEJM 2016; Peyvandi et al, JTH 2017 clinicaltrials.gov NCT02553317 Hercules trial -phase III study

• Start/End date: Sep 2015 – Sep 2017 • 145 patients enrolled • Caplacizumab meets primary and key secondary endpoints

Preliminary analysis of HERCULES trial data confirmed TITAN trial results

Ablynx press release, 2 October 2017 Changing the treatment paradigm

Future standard of care based on three pillars

Daily PEX until confirmed Immunosuppression Caplacizumab platelet normalization (corticosteroids and/or RTX)

Treatement duration Immediate blocking of binding Remove ULVWF and anti- of vWF to platelets ADAMTS13 autoantibodies Protection against Replace functional ADAMTS13 microvascular thrombosis, organ damage and long-term consequences (loss of memory, Suppress over-activity of headache…) immune system Reduction in exacerbations Reduction in days & complications of PEX rADAMTS13

• rADAMTS13 (BAX930, now SHP655) was shown to overcome ADAMTS13 inhibitors and restore ADAMTS13 activity in vitro and in a rat model of acquired TTP

• rADAMTS13 may be a promising candidate for further exploration in treating acute episodes of acquired TTP

Plaimauer et al, JTH 2011 & JTH 2015, Tersteeg et al, ATVB 2015 REMISSION PHASE Risk of TTP recurrence

• Almost 1/3 of patients who survive the first acute episode of TTP will relapse after one month – many years and will develop a chronic recurrent form of TTP

Zhan H et al Transfusion, 2010

Risk factors for recurrence? • Low ADAMTS13 levels <10% and presence of anti-ADAMTS13 antibodies • Infections • Surgery • Some drugs (estrogens, thyenopiridines, quinine, etc.) • Ferrari et al, Blood 2007; Jin et al BJH, 2008; Peyvandi et al, Haematologica 2008; Ferrari et al, JTH 2009; Yang et al, ISTH 2015; Mancini et al, Eu J Inter Med 2017 Remission phase management

WAIT and SEE vs Preventive Therapy Preventive therapy: treatment options

• Rituximab – Patients treated with RTX during acute phase had a significantly reduced relapse rate compared with the hystorical control (10 vs 57%) (Scully et al, Blood 2011) – Relapse-free survival was longer in patients treated with preemptive infusion of RTX during remission phase (Hie et al, Blood 2014) Preventive therapy: rituximab

• In TTP patients with ADAMTS13 levels < 10% during remission, relapse-free survival was longer in patients treated with rituximab than in untreated ones (p=0.049)

• 0 relapses/year among patients treated with rituximab • 0.57 relapses/year among untreated patients (p < 0.01)

Hie et al, Blood 2014 Other immunosuppressive therapy

• Corticosteroide • CSA • Azatiophrine • Splenectomy Improvements in TTP therapy

Moschowitz, Proc N Y Pathol Soc 1924; Burke and Hartmann, JAMA 1959; Bukowski et al, Blood 1977; Byrnes and Khurana, NEJM 1977; Moake et al, NEJM 1982; Furlan et al, Blood 1996; Tsai Blood 1996; Levy et al, Nature 2001; Zheng et al, JBC 2001; Chemnitz et al, American Journal of 2002; Chapin et al, BJH 2012; Shortt et al, NEJM 2013; , Li et al, Transfusion 2014; Scully et al, ASH congress 2016; Peyvandi et al, NEJM 2016 Conclusions

• From plasma infusion in the 70’ to caplacizumab nowadays: the unraveling of pathogenic mechanisms underlying this disease resulted in the development of more and more targeted therapies • Despite recent advances, TTP still remains a severe and unpredictable disease • Many open questions on TTP still remain: – Which is the optimal treatment in the acute phase to prevent exacerbation and reduce organ damage (kidney, heart, brain) with less chronic sequelae?

– Which patients will relapse? Which are the predictive markers? – Which is the best treatment during remission? Acknowledgments

Angelo Bianchi Bonomi and Thrombosis Center and Fondazione Luigi Villa